1. Field of the Invention
The present invention relates to a human growth hormone agent for adults, more specifically to a human growth hormone agent for replacement therapy for growth hormone-deficient adult patients.
2. Description of the Related Art
A human growth hormone is a hormone which is continuously secreted from the pituitary gland not only during a growth period but also all through life in a normal human. The human growth hormone was first extracted and purified from the human pituitary gland. The human growth hormone has known to exist in two types: one having a molecular weight of about 22,000 (hereinafter referred to as 22-kilodalton human growth hormone) and the other having a molecular weight of about 20,000 (hereinafter referred to as 20-kilodalton human growth hormone). It has been reported that the 20-kilodalton human growth hormone has a growth promoting activity equivalent to that of the 22-kilodalton human growth hormone but has a weaker activity in inducing glucose intolerance than the 22-kilodalton human growth hormone so that it may be less diabetogenic (Lewis U. J., et al: Endocrinol. Japan 34 73-85, 1987). Accordingly, effectiveness of the 20-kilodalton human growth hormone as a therapeutic agent for growth hormone deficiency has attracted attention (Hisao Seo, et al: Igaku no Ayumi 165 247-251, 1993).
However, in spite of the development in recombinant DNA technology, production of the 20-kilodalton human growth hormone was extremely difficult and the amount of expression of the 20-kilodalton human growth hormone having an additional Met at the N-terminal in an intracellular expression method was {fraction (1/20)} that of the 22-kilodalton human growth hormone (Miyamoto, et al: International Symposium on GRF; Growth Hormone and Somatomedin Program and Abstracts 28 1-2, 1986). In manufacturing proteins, it is known that generally, higher-order structures in the cells are not properly formed in the intracellular expression method, so that a refolding process is required. However, since unlike the 22-kilodalton human growth hormone, there is no international standard product for this 20-kilodalton human growth hormone, it is difficult to evaluate the appropriateness of the structure attained by the refolding process by comparison with a standard product. Furthermore, this 20-kilodalton human growth hormone is highly hydrophobic, which makes purification process difficult and substantially defies a secretion method for the production of an authentic 20-kilodalton human growth hormone having the amino acid sequence identical to that of the 20-kilodalton human growth hormone derived from the pituitary gland.
Thus, such a poor availability of the authentic 20-kilodalton human growth hormone has been an obstacle to study its potentiality as a medicament (Fukashi Matsuzaki: Igaku no Ayumi 165 243-246, 1993).
Biological properties of the human growth hormone, which are the foundation of its potentiality as a medicament, will be described as follows:
A growth hormone is associated with various events in the body, such as lipolysis, protein anabolism and osteogenesis. Therefore, in growth hormone-deficient patients, a lipolytic activity, an insulin-like growth factor I (hereinafter referred to as IGF-I) secreting activity, a protein synthesis stimulating activity, a bone metabolizing activity or the like are being decreased. Among them, the decrease in lipolytic activity is one of the factors to cause accumulation of subcutaneous fat or visceral fat, which results in marked obesity. Thus, in growth hormone-deficient patients, a complication with hyperlipemia is known to occur in many cases and the morbidity in circulatory disorders such as arteriosclerosis and the mortality from cardiovascular diseases are reported to be high. It has also been reported that since IGF-I is associated with various physiological events in the body, the decrease in the serum IGF-I concentration due to the reduced IGF-I secreting activity causes various disorders. Furthermore, it has also been reported that the decrease in the protein synthesis stimulating activity causes a decrease in muscular strength and exercise capacity, which may cause problems in daily activities.
From the fact described above, in growth hormone-deficient patients, it is required to improve body composition, namely to bring body protein content, body fat content and bone density close to normal levels. More specifically, it is required to increase a decreased body protein content and bone density and to decrease an increased body fat content. To decrease the body fat content here implies suppression of body fat accumulation, stimulation of body fat hydrolysis and the like so that the stimulation of body fat hydrolysis is not the only cause of the decrease in the body fat content. Therefore, the stimulation of body fat hydrolysis is one of the factors to improve the body composition.
Some activities related to the 22-kilodalton human growth hormone in growth hormone-deficient patients are conventionally known as follows:
As a replacement therapy for growth hormone-deficient patients, administrations of the 22-kilodalton human growth hormone produced by a recombinant DNA technique to the patients have been tried; with regard to the above-mentioned improvement of the body composition, it was reported that an administration of the 22-kilodalton human growth hormone over a period of 3 years reduced the body fat content to normal level (Jorgensen, J. OL., et al: European Journal of Endocrinology 130 224-228, 1994) and an administration of the 22-kilodalton human growth hormone over a period of 6 weeks increased the serum IGF-I concentration to a normal level, and improved the body nitrogen level, which implies the body protein level, bringing it close to normal (Bengtsson, B. A., et al: J. Clin. Endocrinol. Metab. 76 309-317, 1993). However, with regard to adults other than growth hormone-deficient patients, no knowledge is available since no administrative experiment has been conducted.
On the other hand, there is concern that the administration of human growth hormones to adults tends to induce glucose intolerance. In fact, it was indicated that the administration to growth hormone-deficient adult patients induced not frequent but serious abnormalities in glucose metabolism (Beshyah, S. A., et al: Endocrinology and Metabolism (suppl. B), 32, 1994).
It has been difficult to obtain the authentic 20-kilodalton human growth hormone. As a result, in most experimental studies regarding physiological properties of the 20-kilodalton human growth hormone to date, either a purified product prepared from the pituitary gland or a non-authentic recombinant product having a methionine at the N-terminal (hereinafter referred to as Met-type 20-kilodalton human growth hormone) which is prepared by a recombinant gene technique, had been used. Physiological properties obtained from the experiments using these products are described as follows:
A growth promoting activity of the 20-kilodalton human growth hormone, which was extracted and purified from the pituitary gland, was reported to be the same as that of the 22-kilodalton human growth hormone (Lewis, U. J., et al: J. Biol. Chem. 253 2679-2687, 1978). As to affinity to adipocytes and lipolysis stimulating activity, the following is known. The affinity of the 20-kilodalton human growth hormone, which is extracted and purified from the pituitary gland, to adipocytes is as markedly low as 3% of that of the 22-kilodalton human growth hormone (De Meyts, P., et al: J. Biol. Chem. 262 11071-11079, 1987). On the other hand, as to the lipolysis stimulating activity, which is important in the administration to growth hormone-deficient adult patients, a serum free fatty acid concentration was increased by 40% due to the lipolysis stimulation in vivo with the 22-kilodalton human growth hormone, while no increase was observed with the 20-kilodalton human growth hormone which was extracted and purified from the pituitary gland (Lewis, U. J., et al: Biochem. Biophys. Res. Commun. 91 778-782, 1979). Furthermore, the 20-kilodalton human growth hormone, which was extracted and purified from the pituitary gland, had no lipolysis stimulating activity in vitro (Lewis, U. J., et al: Metabolism 4 237-243, 1985).
Thus, the 20-kilodalton human growth hormone was conventionally considered to have no or reduced lipolysis stimulating activity as compared to the 22-kilodalton human growth hormone. This fact defies anticipation that the 20-kilodalton human growth hormone might have an advantage which is expected from its low potential to induce glucose intolerance in the administration to adults, particularly to growth hormone-deficient adult patients and thus possibly denies its effectiveness in the above-mentioned lipolysis stimulation and body composition improvement in the administration to the growth hormone-deficient adult patients.
As to the serum IGF-I concentration increasing activity, the following is known. The serum IGF-I is known to be released primarily from the liver by binding of growth hormones to liver receptors (xe2x80x9cIGF-growth hormone and its related peptidesxe2x80x9d by Kazuo Shizume, 132-136, 1992); however, the affinity of the 20-kilodalton human growth hormone, which is extracted and purified from the pituitary gland, to the liver receptors is known to be 3-12% in rats and 8-20% in rabbits of that of the 22-kilodalton human growth hormone, respectively (Sigel, M. B., et al: Endocrinology 108 1600-1603, 1981); in humans, the affinity to the liver receptors is known to be almost none with the 20-kilodalton human growth hormone, which is extracted and purified from the pituitary gland, and with the Met-type 20-kilodalton human growth hormone (Baumann, G., et al: Molecular and Cellular Endocrinology 73 11-14, 1990).
Accordingly, it was suggested that the 20-kilodalton human growth hormone has none or low activity for IGF-I producing in the liver and releasing from the liver. Yet, there was a report that the 20-kilodalton human growth hormone, which was extracted and purified from the pituitary gland, brought a serum IGF-I concentration similar to that with the 22-kilodalton human growth hormone, when administered to hypophysectomized rats (Spencer, E., et al: Endocrinol. 101 1301-1302, 1981). However, it was pointed out that in purifying the 20-kilodalton human growth hormone from the pituitary gland, a complete separation of the 20-kilodalton human growth hormone from the 22-kilodalton human growth hormone is difficult because of their similarity in physicochemical properties and therefore the 20-kilodalton human growth hormone, which is extracted and purified from the pituitary gland, used for experiments to date was mixed with the 22-kilodalton human growth hormone, which might have affected results of the experiments (Baumann, G., et al: Molecular and Cellular Endocrinology 73 11-14, 1990). Consequently, sufficient knowledge on the basis of studies using a pure 20-kilodalton human growth hormone has not been attained.
As mentioned above, as to the authentic 20-kilodalton human growth hormone, almost no knowledge had been attained in regard to its activity in improving the body composition, stimulating lipolysis or increasing the serum IGF-I concentration.
The subject of the present invention is to solve the problem in providing a human growth hormone agent for adults, which is effective in improving the body composition, stimulating lipolysis and/or increasing the serum IGF-I concentration and accordingly, an object of the present invention is to provide a human growth hormone agent for replacement therapy for adults, particularly for growth hormone-deficient adult patients, which contains an authentic 20-kilodalton human growth hormone as an active component and has a low activity in inducing glucose intolerance.
In order to solve the above-mentioned problem, the present inventors established a process for the production of an authentic 20-kilodalton human growth hormone by a recombinant DNA technique, prepared a pharmaceutical preparation containing the authentic 20-kilodalton human growth hormone and conducted various studies using the resultant human growth hormone agent. As a result, the present inventors novelly found that the authentic 20-kilodalton human growth hormone has significant activities in improving the body composition, stimulating lipolysis and/or increasing the serum IGF-I concentration and furthermore reconfirmed that the authentic 20-kilodalton human growth hormone is less diabetogenic than the 22-kilodalton human growth hormone.
The above-mentioned knowledge on the authentic 20-kilodalton human growth hormone thus obtained could not be predicted from the conventional knowledge on the 20-kilodalton human growth hormone, which is extracted and purified from the pituitary gland, or the Met-type 20-kilodalton human growth hormone. Namely, the knowledge that the authentic 20-kilodalton human growth hormone has the activity to stimulate lipolysis, which is one of the factors to improve the body composition, could not be predicted from results of experiments using the 20-kilodalton human growth hormone which was extracted and purified from the pituitary gland. Furthermore, the knowledge that the pure authentic 20-kilodalton human growth hormone without a 22-kilodalton human growth hormone has the capability to increase the serum IGF-I concentration as did the 22-kilodalton human growth hormone could not be readily predicted from the conventional knowledge that the 20-kilodalton human growth hormone has a low affinity to the liver receptors (Baumann, G., et al: Molecular and Cellular Endocrinology 73 11-14, 1990). The present inventors have now found that the above-mentioned authentic 20-kilodalton human growth hormone has significant activities in improving the body composition, stimulating lipolysis and increasing the serum IGF-I concentration so that the usefulness of the 20-kilodalton human growth hormone as a medicament for adults is confirmed.
The present inventors found that the authentic 20-kilodalton human growth hormone has excellent capabilities in improving the body composition, stimulating lipolysis and increasing the serum IGF-I concentration, contrary to the conventional knowledge so far reported on the 20-kilodalton human growth hormone.
From the knowledge mentioned above, this agent can be used as a human growth hormone agent for adults, particularly as an excellent human growth hormone agent in replacement therapy for growth hormone-deficient adult patients.